Determination of Multi-ResidueTetracyclines and their Metabolites in Milk by High Performance LiquidChromatography - Tandem MassSpectrometry

Abstract

Tetracyclines are probably the most frequently used antibiotics in animal husbandry.

In this paper, a high performance liquid chromatography tandem mass spectrometric

(HPLC /MS/MS) method is developed for the simultaneous determination of 10

antibiotic residues: minocycline, 4-epioxytetracycline, 4-epitetracycline, tetracycline,

4-epichlortetracycline, demeclocycline, chlortetracycline, methacycline, doxycycline,

oxytetracycline in milk and animal tissues. In the method, Agilent’s novel solid phase

extraction cartridge and a reversed phase Agilent ZORBAX RX C8 column (5 µm, 150

mm × 2.1 mm) are used for purification and separation. The limit of detection (LOD) is

between 0.5 and 10.0 µg/kg and the limit of quantitation (LOQ) is less than 50 µg/kg.

The linearity is obtained from 5 to 1000 µg/kg. Overall recoveries are between 76.4%

and 101% with a relative standard deviation (RSD, n = 6) less than 8.4%. The method

is rapid, sensitive, convenient and robust, and can be used to simultaneously confirm

multi-residues of tetracyclines and their metabolites in milk.

Authors

Yanyan Fang, Hao Zhai, and Yun Zou

Agilent Technologies (Shanghai), Co.

412 Yin Lun Road 200131

China

Jerry Zweigenbaum

Agilent Technologies, Inc.

2850 Centerville Road

Wilmington, DE 19809

USA

Application NoteFood

2

Introduction

Antibiotics are used worldwide to control bacterial infectionand promote healthy farm animals for milk production.Tetracyclines are broad-spectrum antibiotics, so they arewidely used. However, it is undesirable to have them in themilk supply.

FDA's regulations for tetracyclines including oxytetracyclineand chlortetracycline are set to provide an acceptable dailyintake (ADI) and a tolerance for residues in milk. The ADI fortotal residues of these compounds is 25 micrograms per kilo-gram of body weight per day. Sixty percent (60 %) of the ADIis reserved for milk and 40 % for edible tissues. Based on theADI, a tolerance of 300 ppb is set for the sum of residues ofthe tetracyclines including chlortetracycline, oxytetracycline,and tetracycline in milk. With the establishment of a toler-

ance of 300 ppb for the sum of residues of tetracyclines, a tol-erance of 300 ppb for each of the three tetracyclines is alsoaccepted.

In the EU, the maximum residue limit (MRL) for antibiotics isestablished according to (EEC) 2377/90, and for tetracyclinesin milk is at 100 µg/kg (100 ppb). In China, the GovernmentStandard (GB/T 21317-2007) also establishes the method fordetermination of these compounds in milk and animal tissues.This regulation took effective April 1, 2008.

The purpose of this study is to develop a method for theAgilent 6410 LC/MS/MS to determine the presence of tetra-cyclines and their metabolite residues in milk. The method israpid and easy to use. The tetracyclines and their metabolitesare given in Table 1.

Table 1. The Compounds in this Study

No. Name CAS No. Structure

1 Minocycline 10118-90-8

2 Oxytetracycline 6153-64-6

3 Tetracycline 60-54-8

4 Demeclocycline 127-33-3

OO

NN

OOH OHOH

OH

CH3

NH2

CH3H3C H3C

H H

OH

OH OHOH

OH

NH2

CH3

H3C NH3C

OO O

C

HH

OH OH

OH

OH

OH

NH2

CH3H3C

H3C

OO

O

NH

H

OH

OH

OH

NH2

CH3H3C

OO

N

OOO OOH OH

ClHH

(Continued)

3

OH OHOH

OH

HOCl

NH2

CH3

CH3

H3C

OO OO

NH

H

OO OOH OHOH

OH

OH

NCH3

CH2

NH2

H3C

HH

NH2

OO OOH OHOH

OH

OH

CH3

CH3

H3CH2O

HN

H

CH3

OO OOH OHOH

OHHO

CH3

NH2

H3CN

H

No. Name CAS No. Structure

5 chlortetracycline 57-62-5

6 methacycline 914-00-1

7 doxycycline 564-25-0

88 4-epitetracycline 64-75-5

9 4-epi oxytetracycline 35259-39-3

10 4-epichlortetracycline 14297-93-9

N

O

OO OHOH

OHOH

HO

NH2

H3CCH3H3C

OH OHOHOH

CH3H3C

H2N

OHH3C Cl

OO

O

N

Table 1. The Compounds in this Study

4

Experimental

Reagents and ChemicalsWater and methanol are HPLC grade, and they, along withformic acid were all purchased from Fluka. The standardswere purchased from Sigma-Aldrich.

Instrument SettingsTable 2. LC/MS/MS Conditions

HPLC

Column ZORBAX RX-C8, 2.1 mm × 150 mm, 5 µm (p/n 883700-906)

Flow rate 0.3 mL/minMobile phase A: Water/ 0.1 % Formic Acid

B: MethanolGradient 0–10 min, B from 5% to 30%

10–12 min, B from 30% to 40%12.5–18 min, B 65%18.5–25 min, B 95%25.5 min, B 5.0%

Total run 28 minPost time 5 minTemp 30 °Cinjection 5 µL

MS Source settings

Source ESIIon polarity PositiveDrying Gas temp. 350 °CDrying gas flow rate 10 L/minNebulizer 45 psiVcap 4000V

Precursor Product Rt.Name Frag. ion ion CE (min)

Minocycline 120 458 352 35441 20 8.58

4-Epitetracycline 120 445 410 20427 10 8.60

4-Epioxytetracycline 120 461 426 20444 15 9.47

Tetracycline 120 445 410 20427 15 9.90

Oxytetracycline 120 461 426 20443 10 9.95

Demethylclocycline 120 465 430 25448 15 11.25

4-Epichlortetracycline 120 479 444 22462 15 11.59

Chlortetracycline 120 479 444 22462 15 12.95

Methacycline 120 443 381 25426 15 13.98

Doxycycline 120 445 154 30428 15 14.08

MRM Setting

Sample PreparationExtraction:1. Weigh a 5 g-milk sample (accurate to 0.01 g) into a 50-mL

colorimetric tube, and dissolve with 0.1 mol/L Na2EDTA-Mcllvaine buffer solution and bring volume to 50 mL.

2. Vortex for 1 min and ultrasonicate the extract in an ice water bath for 10 min.

3. Transfer the sample to a 50-mL polypropylene centrifuge tube and cool to 0 °C ~ 4 °C.

4. Centrifuge the sample at a speed of 5000 rpm for 10 min (below 15 °C).

5. Filter with fast filter paper.

Purification: 1. Accurately draw 10 mL of the extract (equivalent to 1 g

sample) and put it through the SampliQ OPT cartridge (p/n 5982-3036) at a speed of 1 drop/s.

2. After it elutes completely, clean the cartridge with 3 mL water adjusted to pH 4.5 with trifluoroacetic acid and then discard the entire effluent.

3. Under a negative pressure below 2.0 kPa, drain the cartridge for 5 min.

4. Elute with 10 mL of 10 mmol oxalic acid in methanol.

5. Collect the eluent and dry with nitrogen below 40 °C.

6. Dissolve the residue with 1.0 mL of the initial mobile phase.

7. Filter with a 0.45-µm filter membrane and inject.

Results and Discussion

Optimization and Separation

Fragmentor and Collision Energy (CE) optimization

It is well known that the LC/MS/MS QQQ is the best tool toidentify, confirm and quantify target analytes in food matrices.In order to get the best response, only two parameters needto be optimized for each compound on this instrument, thefragmentor and the collision energy. The correct fragmentorvoltage allows the highest transmission of the precursor ioninto the mass analyzer. The correct collision energy providesthe highest intensity of quantitation of the qualifier production.

5

0.4

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1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2+ TIC Scan erjiaan_Frag_01.d 1 1 2 2 3 3 4 4 5 5 6 6

Counts vs. acquisition time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2

×108

Figure 1. Optimization of fragmentor voltage for minocycline from 60-160 by steps of 20 V.

sor ion, and optimize the fragmentor voltage by steppingthrough the increments that the user selected for one injec-tion. The program then selects the voltage producing thehighest intensity for the precursor ion. For tetracycline, this isshown in Figure 3.

The program then performs a product ion scan on a secondinjection of the sample, and chooses the four most prevalentproduct ions. It reinjects the sample again and performsMRMs of each ion collected with collision energies in incre-ments covering the range of voltage selected by the user. Thecollision energy that generates the maximum signal for eachproduct ion is then automatically determined and can bestored in the database. The data from this collision energyoptimization for tetracycline is shown in Figure 4 along withthe ion breakdown curve shown in Figure 5. Compounds withproduct ions can be imported directly into the users’ acquisi-tion method.

One method of optimization is to inject the sample multipletimes at the different fragmentor voltages set within seg-ments of a single run. This is shown in Figure 1 for minocy-cline. For this compound, there is a small increase in detec-tion as the voltage is increased. Collision energy is optimizedin the same way and the results for tetracycline is shown inFigure 2.

Recently, Agilent introduced the "Optimizer" program thatautomatically determines the optimum fragmentor voltageand collision energy and stores the results in the OptimizerDatabase. Using this program and flow injection with or with-out a column, the user enters the compounds to be optimizedand their molecular formulas. The nominal mass of the com-pound is automatically calculated from the formula. The userthen specifies the adducts expected for positive and negativemodes, the low mass cutoff, any ions to be excluded, and themethod to be used (mobile phase conditions etc.). Once start-ed the program will inject the sample, determine the precur-

6

×105

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ESI EIC:3 (444.7-445.4) SIM Frag = 90.0V tetracycline_OPTIMIZE_MS2SIM.d

Counts vs. acquisition time (min)

0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.36 0.38 0.4 0.42 0.44 0.46 0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62

+ESI EIC:1 (444.9-445.4) SIM Frag = 50.0V tetracycline_OPTIMIZE_MS2SIM.d +ESI EIC:2 (444.7-445.5) SIM Frag = 70.0V tetracycline_OPTIMIZE_MS2SIM.d +ESI EIC:4 (444.7-445.5) SIM Frag = 110.0V tetracycline_OPTIMIZE_MS2SIM.d +ESI EIC:5 (444.6-445.7) SIM Frag = 130.0V tetracycline_OPTIMIZE_MS2SIM.d +ESI EIC:6 (444.1-445.8) SIM Frag =150.0V tetracycline_OPTIMIZE_MS2SIM.d

Figure 3. Single injection automatic determination of fragmentor voltage for tetracycline using the Optimizer program.

×105

0.2

0.6

1

1.4

1.8

2.2

2.6

3

3.4

3.8

4.2

4.6

5

5.4

5.8

6.2

6.6

7

+ TIC MRM (** & **) sihuansuCE_01.d 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

Counts vs. acquisition time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4

Figure 2. Manual collision energy optimization of tetracycline.

7

×104

Counts vs. acquisition time (min)

0.2 0.22 0.24 0.26 0.28 0.3 0.32 0.34 0.36 0.38 0.4 0.42 0.44 0.46 0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

+ESI MRM Frag=82.0V CID@25.0 (445.2 & 154.1) tetracycline_OPTIMIZE_MRM.d

Figure 4. Single injection automated collision energy determination using Optimizer program for tetracycline.

Figure 5. Ion breakdown profile for tetracycline as determined by theOptimizer program.

8

SeparationSample preparation and separation of tetracycline, chlortetra-cyline and oxytetracycline is important. The challenge in sep-arating these kinds of compounds is that they easily degradeunder conditions of weak acid, strong acid, strong base, andheat converting the diasteriomer to its diaxial epimer.

The typical process is shown below with tetracycline:

Figure 6. The degradation of tetracycline to 4-epitetracycline.

9

Tetracyclines and their degradants are diasteriomers with thesame formula and the same fragment ions are formed inMS/MS. Therefore, they have the same precursor ions, quali-tative ions, and quantitation ions. In order to identify and con-firm them in the Rapid Resolution liquid chromatograph(RRLC), separation is important for this analysis. Using theAgilent ZORBAX Rx-C8, 2.1 mm × 150 mm, 5-µm particle sizecolumn and a simple gradient, the three epimer pairs of thesecompounds are well separated. This is shown with the reten-tion times given in Table 2. Figure 7 shows the graphic repre-sentation of the separation of tetracycline and its epimer.

×105

×105

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4+ TIC MRM (** & **) chaxiangsihuansu_01.d

1 1

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

+ TIC MRM (** & **) sihuansu_01.d

1 1

Counts vs. acquisition time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

a. 4-epitetracycline

b. Tetracycline

Figure 7. The separation of tetracycline and its degradation product 4-epitetracycline.

Linearity, LOD and LOQLinearity, LOD and LOQ were evaluated in both solvent and amilk matrix. The results are given in Table 3 and show that lin-earity is similar for both solvent and milk matrix and generallyprovide greater than 0.99 coefficient of variance. The tetracy-clines do not ionize well with electrospray but the limits of

10

detection (LOD) for each are still in the low pictograms on-column. The limit of quantitation is typically set at a signal tonoise (S/N) of 10:1 but we report twice that in the solvent.The graphic representation of the calibration curve forminocycline is shown in Figure 8.

Table 3. Quantitative Performance of Tetracyclines in Solvent and Milk Matrix

Standards in solvent* Standards in Milk matrix*

Name R2 LOQ LOD R2 LOD(S/N=20) (S/N=3) (S/N=3)pg on column pg on column pg on column

Minocycline 0.999 41.5 6.2 0.990 16.3

4-epitetracycline 0.991 10.8 1.6 0.994 8.7

4-epioxytetracycline 0.996 14.7 2.2 0.996 12.8

Tetracycline 0.998 9.4 1.4 0.994 10.2

Oxytetracycline 0.996 10.7 1.6 0.991 8.6

Demethylclocycline 0.999 22.8 3.4 0.993 8.1

4-epichlortetracycline 0.986 38.2 5.7 0.987 11.9

Chlortetracycline 0.986 8.1 1.2 0.994 7.6

Methacycline 0.999 20.8 3.1 0.994 12.3

Doxycycline 0.999 32.2 4.8 0.995 11.2

Note: *The calibration curve range is from 1 ppb-1 ppm with injection volume of 5 uL

×105

jiaxitumeisu - 7 Levels, 7 Levels Used, 21 Points, 21 Points Used, 0 QCs

Concentration (µg/L)

-50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050

Res

pons

es

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

y = 143.4386 * × - 442.6564

R2 = 0.99960865

Figure 8. Tetracycline calibration curve from 1 ppb to 1000 ppb.

11

Recovery and RepeatabilityThe recovery and repeatability of the method was evaluatedand the results shown in Table 4. All recoveries were greaterthan 80 % which is generally accepted as sufficient. In addi-tion the precision, as shown in the table, is 5 % or better forthe lower concentration and less than 2 % for the higher con-

centration. Ion ratios for confirmation are a very importantperformance criterion and these results show excellentrepeatability. A graphic representation of the ion ratios formethacycline is shown in Figure 9. The ratios combined withmatching retention time provide the necessary information forconfirmation.

Table 4. Recovery and Repeatability in Milk Matrix

Recovery in milk RSD % RSD % Recovery in milk RSD % RSD %(Conc. 50 ppb (Signal response (Ion ratio (Conc. 100 ppb (Signal response (Ion ratio

Name n=6) n=6) n=6) n=6) n=6) n=6)

Minocycline 96.5 4.9 2.1 101.4 1.6 1.0

4-epitetracycline 89.2 3.8 1.5 96.3 1.6 0.9

4-epioxytetracycline 84.4 5.4 1.3 88.2 0.9 0.6

Tetracycline 86.1 2.5 1.2 90.7 1.1 1.2

Oxytetracycline 77.6 3.8 1.6 82.5 1.2 0.9

Demethylclocycline 79.2 2.0 3.1 84.7 0.9 0.6

4-epichlortetracycline 76.4 5.5 5.4 84.3 1.1 0.5

Chlortetracycline 94.3 4.5 1.5 100.9 1.8 1.1

Methacycline 86.3 1.0 1.9 91.2 1.2 0.8

Doxycycline 78.7 3.6 6.7 82.4 1.0 0.8

×102×102

Acquisition time (min)12 13 14 15 16

Rel

atie

ve a

bund

ance

(%)

_0.1

_0.050

0.050.1

0.150.2

0.250.3

0.350.4

0.450.5

0.550.6

0.650.7

0.750.8

0.850.9

0.951

1.051.1

1.151.2

1.25

443.0 -> 426.0, 443.0 & 381.0Ratio = 12.7

+ MRM:10 (7.846-9.272 min, 22 scans) (445.0 & ...

Mass-to-charge (m/z)150 200 250 300 350 400 450

Count

s

00.25

0.50.75

11.25

1.51.75

22.25

2.52.75

33.25

3.53.75

44.25

4.54.75

55.25

5.55.75

410.0

154.0

445.0

×104+ MRM (443.0 & 426.0) std11-r002.d

Acquisition time (min)12 13 14 15 16

Cou

nts

_0.1

_0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

114.021

Figure 9. Shows the ion ratios for qualifier ion and the quantitation ion of methacycline.

12

Study of Ion SuppressionIn general, tandem MS can remove chemical noise to get a"clean" spectrum even in dirty and complex food matrices.However, the matrix may contain components that suppressthe ionization of the analyte. Figure 10 shows the comparisonof the response of methacycline and tetracycline in solventand milk. The difference in the slope of each curve demon-strates the suppression effect of the milk matrix.

Because of the strong suppression observed, using the exter-nal standard method (ESTD) for calibration, matrix matchedstandards should be prepared in antibiotic-free milk, or milkknown to not contain the analytes. In this way, the calibrationcurve is generated with the same matrix effects as the sam-ples.

0

400000

800000

1200000

1600000

0 500 1000 1500

Methacycline

0

20000

40000

60000

80000

100000

120000

140000

160000

0 200 400 600

1

2

1

2

Tetracycline

Figure 10. Ion suppression of two of the tetracyclines in milk; 1) response in solvent, 2) response in milk.

13

Conclusions

The results of this work show that the Agilent 6410 triplequadrupole LC/MS System is a robust, sensitive, and repeat-able instrument for the study of tetracyclines residues in amilk matrix. In China, the government standard requirement(GB/T 21317-2007) sets the detection limit at 50 ppb with a100 µL injection. This method easily meets these require-ments. Additionally, these types of antibiotics readily degradeunder the conditions of weak acid, base etc. The preparationmethod used here avoids this reaction, allowing the LCmethod to separate these isomers for reliable confirmationand quantitation. Finally, ion suppression is considered for theLC/MS/MS method when comparing different compounds inthe same matrix to their response in solvent. Using the ESTDmethod, the preparation of a matrix-matched calibration curveis necessary to obtain accurate results, even though therecoveries measured for the sample preparation are betterthan 80%.

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